Since 1990, technology called microarray has started to be developed and used in the biological, medical, and pharmaceutical fields. A microarray is obtained by immobilizing several tens to several tens of thousands of probes onto a substrate made of glass, plastic, or the like, and is for detecting, with fluorescence or the like, by applying a sample (target) labeled with fluorescent molecules or the like to this substrate, binding reaction between the probes and the sample. Microarrays enable comprehensive measurement at one time and are expected to become essential to personalized medicine in the future.
Conventionally, a DNA microarray (hereinafter, “DNA chip”) obtained by immobilizing DNAs as probes onto a substrate, a protein microarray obtained by immobilizing proteins as probes onto a substrate, a tissue microarray obtained by immobilizing a number of small specimens as probes onto a substrate, a compound microarray obtained by immobilizing a number of low-molecular compounds as probes onto a substrate, and the like, have been known.
Among them, the DNA chip has been put into practical use at the most advanced level, studies have been performed actively to search for genes related to diseases and perform examination and diagnosis by using those genes, and some of these have been put into practical use.
The DNA chip, which is one mode of a microarray, will be described in detail below.
The DNA chip is obtained by spotting (immobilizing), in a grid form, DNAs onto a substrate made of glass, resin, or the like. On the DNA chip, single stranded DNAs (DNA probes) are spotted as probes that are able to specifically react with a DNA sample to be labeled. The DNA probes to be used are those with known sequences. An optically detectable luminescent or fluorescent mark is added to the DNA sample (single stranded DNA) to be analyzed, the DNA sample having an unknown sequence. As a result, when the DNA sample with the unknown sequence to be analyzed is caused to flow onto the DNA chip, if the sequence of the DNA sample is in a complementary relation with a sequence of a DNA probe, the DNA probe and the DNA sample bond to each other to form a double stranded DNA. Therefore, when all of the DNA sample that has not bonded to any of the DNA probes is washed out, the DNA sample to be determined that remain on the DNA chip is made luminescent, and this luminescence is read by a reading device (scanner), the state of any double stranded DNAs is able to be observed as an image. That is, by analyzing distribution of luminescent marks on the DNA chip, presence of the gene to be sought for, whether or not a certain gene has been expressed, or the degree of the expression is able to be analyzed. As described above, by forming a DNA probe set having known sequences on a DNA chip and mounting the DNA probes having different sequences from one another on the DNA chip, genetic alteration, an expression amount of a gene, and the like are able to be detected.
FIG. 13 illustrates a series of procedural steps of DNA chip analysis in detail.
In a preprocessing step illustrated in FIG. 13, unknown DNAs contained in a DNA sample extracted from a specimen are amplified and a fluorescent mark (for example, Cy3, Cy5, or the like) is added to the DNAs (Step S201).
Next, in a hybridization step, the DNA sample added with the fluorescent mark is dropped onto a substrate of a DNA chip mounted with a number of types of DNA probes. The DNA sample bonds to the spotted DNA probe to form a double strand if the DNA sample is in a complementary relation with the spotted DNA probe (Step S202).
Next, in a washing step, the hybridized DNA chip is washed with a predetermined washer fluid (Step S203). Thereby, all of the DNA sample that has not bonded to the DNA probes arranged in a grid form is washed out.
Subsequently, the washed DNA chip is scanned by irradiation with light (Step S204). In the scanning step, the DNA chip is irradiated with laser light having a wavelength suitable to excite the fluorescent mark and fluorescence from the DNA sample bonded (hybridized) to the respective DNA probes is acquired as electric signals. Thereby, amounts of luminescence of the fluorescent mark added to the DNA sample bonded to the respective spotted DNA probes (genes) are measured and fluorescence image data, on which an analyzing process is to be performed based on the amounts of luminescence, are acquired.
In an analyzing step, a fluorescence intensity of each spot is calculated by using a template for the acquired fluorescence image data and various types of analyses are executed (Step S205).
FIG. 14 illustrates an example of a DNA chip 100 to be used in DNA chip analysis. The DNA chip 100 illustrated in FIG. 14 has a rectangular plate-like form having a concave and convex shape. The DNA chip 100 has a plurality of blocks 101 formed by a plate face thereof being divided in a grid form. On each of the blocks 101, a plurality of spots 102 are formed, which are each provided in an approximately column shape or truncated cone shape, immobilize DNA probes corresponding to individual genes, and are arrayed, with a predetermined number thereof in a row direction and a predetermined number thereof in a column direction, in a matrix form. Further, the plurality of blocks 101 are formed on a bottom portion of a concave portion 103 that has been notched into a rectangular column shape. The DNA probes arranged on the spots 102 correspond to genes, which have base sequences that have been already decoded and which are different from one another, and their arrangement positions on the block 101 are determined beforehand.
Further, FIG. 15 illustrates an example of a template to be applied to fluorescence image data of a DNA chip. As illustrated in FIG. 15, the template is divided into a plurality of (for example, 32 in FIG. 15) blocks (corresponding to the blocks 101), and detection areas (corresponding to the individual spots 102 of the DNA chip 100) that are arranged in a matrix form of “m” rows and “n” columns (“22×22” in FIG. 15) are provided on each block.
In the above-mentioned analyzing step, the detection areas on the template provided by an analysis tool are assigned to the individual spots 102 in the read fluorescence image data of the DNA chip (alignment) to calculate fluorescence intensities of the respective spots 102 in the corresponding detection areas. In that case, to execute the analysis accurately, an alignment process needs to be executed accurately such that the individual detection areas of the template are set correctly to the individual spots 102 on the image.
Methods of that alignment include a pattern matching method and a projection method in which alignment is made block by block. Like the technique disclosed in Japanese Laid-open Patent Publication No. 2005-172840, attempts to perform alignment accurately have been made, by using a chip spotted with a fluorescent substance called positive control or with a house-keeping gene contained in any specimen.
Furthermore, like the technique disclosed in Japanese Laid-open Patent Publication No. 2005-024532, a method has been devised, which performs alignment from an image acquired by making an image of a concave and convex shape from reflected light and/or scattered light from a substrate.
However, with any of the typical pattern matching method and projection method in which alignment is made block by block, accurate alignment is unable to be made unless an amount of hybridized sample DNA is large and the spots 102 that emit fluorescence of a sufficient intensity are present by a quarter to approximately a half of the spots 102 on each block 101. Thus, if a sample extracted from a specimen contains a small amount of DNAs, alignment may be unable to be performed accurately in some cases.
In contrast, the method of arranging a fluorescent substance by spotting the fluorescent substance has an advantage that alignment is able to be performed even if spots that emit fluorescence having a sufficient intensity are few, but has problems in that the number of DNAs that are able to be arranged on the spots 102 is reduced and the cost upon manufacturing the chips is increased, for example. Further, when the fluorescent substance is spotted, there is a risk that the fluorescent substance may liberate during the hybridization to contaminate the periphery of the positive control and data may not be able to be acquired.
It could therefore be helpful to provide a detecting method, a microarray analyzing method, and a fluorescence reading device that enable acquirement of an image, from which a height difference of a substrate is accurately detectable.